I. Glutamic Acid Decarboxylase (GAD) and Gamma-aminobutyric Acid (GABA)
The major inhibitory neurotransmitter in the brain is gamma-aminobutyric acid (GABA), (Roberts et al, GABA in Nervous System Function, Raven Press: New York, 1976; McGeer E G, et al, Glutamine, Glutamate, and GABA in the Central Nervous System; Hertz L, Kvamme E, McGeer E G, Schousbal A, eds., Liss: New York, 1983;3-17). The loss of GABA signaling, by a reduction in GABA release, loss of neurons which synthesize GABA, or antagonism of GABA receptors leads to disinhibition, overexcitation. Depending on the specific brain region involved, this loss of signaling may result in epilepsy, movement disorders or other neurological deficits and symptoms.
Gamma aminobutyric acid (GABA) and glutamic acid are two major neurotransmitters involved in the regulation of brain neuronal activity. GABA is the major inhibitory neurotransmitter and L-glutamic acid is an excitatory transmitter (Roberts et al. GABA in Nervous System Function, Raven Press: New York, 1976; McGeer et al. Glutamine, Glutamate, and GABA in the Central Nervous System; Hertz L, Kvamme E, McGeer E G, Schousbal A, eds., Liss: New York, 1983;3-17). GABA is released from dopaminergic cells. An imbalance in the concentration of these neurotransmitters can lead to convulsive states. When the concentration of GABA diminishes below a threshold level in the brain, convulsions result (Karlsson et al., (1974) Biochem. Pharmacol. 23:3053-3061). When the GABA levels rise in the brain the convulsions terminate (Hayashi (1959) supra). In several convulsive disorders there is concomitant with reduced brain GABA levels, a diminished level of glutamic acid decarboxylase (GAD) activity (McGeer et al., GABA in Nervous System Function; Roberts E, Chase T N, Tower D B, eds., Raven Press: New York 1976:487-495; Butterworth et al., (1983) Neurochem. 41:440-447). The concentrations of GAD and GABA vary in parallel (i.e., are positively correlated) because decreased GAD concentration results in lower GABA production.
GABA interacts with a least two receptors, GABA-A and GABA-B. GABA-A receptors have been well characterized and are coupled to chloride channels (Bormann (1988) Trends Neurosci. 11: 112-116). GABA-A receptors are related to ligand gated ion channels belonging to the same superfamily as the nicotrinic receptor for acetylcholine. In contrast, GABA-B receptors are less well understood, although reports describe that the GABA-B receptors are coupled to either calcium or potassium channels (Bormann (1988) Trends Neurosci. 11:112-116 supra).
The majority of neurons in the striatum (caudate-putamen, dorsal striatum; nucleus accumbens, ventral striatum) and in striatal projection regions (the pallidum, the entopeduncular nucleus and substantia nigra reticulata) use GABA as transmitter and express GAD in the synthesis of GABA.
There are two main forms of GAD present in the vertebrate brain, GAD65 and GAD67, which are the products of two separate genes (Bu et al., 1992). Both forms of the protein are co-expressed throughout the brain but differ in their structure, subcellular localization and regulation. These differences suggest the two GAD isoforms may play differing roles in GABA-mediated neurotransmission.
Human GAD65 and GAD67 have been isolated and cloned by Bu et al. (1992) Proc Natl Acad Sci 89:2115-2119. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues (Genbank Accession No. NM000818;M81882), Human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues (Genbank Accession No. NM013445;M81883).
The human GAD proteins are comprised of two distinct domains. The C-terminal domain, which contains the catalytic site and cofactor binding site, is relatively conserved between human GAD65 and GAD67 with 73% identity. The N-terminus, which contains a membrane association domain, is highly divergent with only 23% identity (Bu et al., 1992).
Targeting of GAD65 to the golgi is mediated by a 27 amino acid domain in the N-terminus, which is not present in GAD67. In CHO (Chinese Hamster Ovary) and COS cells, membrane association of GAD67 is dependent on the presence of GAD65, presumably through heterodimer formation (Dirkx R, 1995). Targeting to presynaptic clusters is mediated by a palmitoylated 60 amino acid N-terminal domain of GAD65 (Kanaani et al., 2002).
An immunoprecipitation study determined that 33% of GAD protein in rat brain extract is present as GAD65/67 heterodimers (Kanaani et al., 1999). Similarly, in another study 27% of GAD protein isolated from rat cerebellum was in the form of GAD65/67 heterodimers (Sheikh and Martin, 1996). GAD67 has, however, been found to associate with membranes in GAD65−/− mice, suggesting that axonal targeting and membrane association can occur via a mechanism independent of GAD65 (Kanaani et al., 1999).
Both GAD65 and GAD67 require the presence of the cofactor pyridoxal phosphate (PLP) for enzyme activity (Martin et al., 1991). Half of GAD65 protein occurs in the inactive apoenzyme form without bound PLP whereas GAD67 occurs mostly in the active holoenzyme form (Erlander et al., 1991). This inactive pool of “stored” GAD has been postulated to be available for activation at times of high or sudden demand for GABA.
There are marked differences in the amount and activity of GAD protein in different areas of the rat brain. The amount of GAD65 was found by immunoblotting to be 77-89% of total GAD protein in twelve brain areas analyzed which correlated with total GAD activity (Sheikh et al., 1999).
Although GAD65 is the predominant form of GAD present in rat brain, there is evidence from knockout mouse studies that GAD67 synthesizes the majority of GABA in the brain. GAD67−/− mice do not display defects in brain morphology at birth but die soon after due to a cleft palate (Asada et al., 1997; Condie et al., 1997). GAD activity and GABA content in the cerebral cortex is reduced to 20% and 7% respectively in newborn GAD67−/− mice (Asada et al., 1997). GAD65−/− mice are viable but GABA levels are low for the first two months after birth (Stork et al., 2000). Adult rats display abnormal neural activity with spontaneous seizures and paroxysmal discharges (Kash et al., 1997). They also have increased susceptibility to picrotoxin induced seizures than their wild type litter mates (Asada et al., 1996). From these observations, it is obvious that although GAD65 and GAD67 contribute to a metabolic pool of GABA, their roles with respect to inhibitory neurotransmission are different. It is possible that due to its presence throughout the neurons, predominantly in the holoenzyme form, GAD67 may contribute to the basic requirements of inhibitory neurotransmission. The low saturation of GAD65 by PLP, combined with the subcellular distribution in axon terminals and anchoring to synaptic vesicles suggest that GAD65 may be involved in the prevention of hyperexcitability by its rapid activation and loading of GABA into vesicles for rapid secretion.
II. Neurological and Other Disorders
Diseases such as Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Epilepsy and Alzheimer's disease, have proved difficult to treat. Few, if any therapies, have proved effective in slowing or arresting the degenerative process associated with these diseases.
In Parkinson's Disease (PD), the primary neurochemical disturbance is believed to be the loss of substantia nigra (SN) dopaminergic (DA) neurons. This loss of DA neurons leads to a profound deficit of DA in the projection areas of the caudate and putamen and results in a loss of signaling through dopamine receptors in the postsynaptic neurons. These neurons, via efferents referred to as the direct and indirect pathways, synapse on other cells in the basal ganglia circuitry. Of most relevance to PD, the loss of dopamine receptors in the basal ganglia circuitry leads to loss of drive in the GABAergic inhibitory input to the subthalamic nucleus.
The loss of inhibitory GABAergic drive to the subthalamic nucleus (STN) results in increased activity of the STN which sends excitatory (glutamatergic) afferents to the ventromedial (VM) thalamus, the substantia nigra pars reticulata (SNPR) and a lesser projection to the pars compacta, as well as other cells within the basal ganglia including the globus pallidus. When the concentration of GABA diminishes below a threshold level in the brain, movement disorders and convulsions may result (See e.g., Karlsson et al, (1974) Biochem. Pharmacol 23:3053-3061). GABA synthesis is regulated by glutamic acid decarboxylase (GAD). GAD is present in the brain as two isoforms, GAD65 and GAD67. When the GABA levels rise in the brain the convulsions terminate (See e.g., Hayashi (1959) Physiol. 145:570-578). In convulsive disorders, the reduction in brain GABA levels is often paralleled by a diminished level of GAD (McGeer, et al. GABA in Nervous System Function; Roberts E, Chase T N, Tower D B, eds., Raven Press: New York 1976:487-495; Butterworth et al. (1983) Neurochem. 41:440-447; Spokes et al. (1978) Adv. Exp. Med. Biol. 123:461-473).
Levodopa (L-dopa) has historically been the medication of choice to treat Parkinson's disease. L-dopa is a precursor to dopamine and is able to cross the blood-brain barrier to target the brain. Unfortunately, the response with L-dopa is not sustainable. Most patients develop adverse effects after long-term usage of L-dopa, and often the benefits of treatment wane as the disease progresses.
Other methods for treating Parkinson's disease include transplantation of cells used to repair regions of the brain damaged by neurodegeneration. These cells can be engineered to secrete neuroactive substances such as L-dopa. The procedure typically involves cell transplantation into the striatum. However, cell transplantation is a complicated procedure which requires donor tissue, and there have been reports of mortality associated with this procedure.
Alternative forms of treating Parkinson's disease involve implanting devices for deep-brain stimulation (DBS) in specific regions of the brain. For example, DBS of the STN. These devices are typically electrodes implanted into the STN. The electrode is then stimulated at a desired frequency to reduce the effect of Parkinson's disease. The significance of the STN overactivity is reflected in the success of ablative surgery of the STN in both animal models of Parkinson's disease, as well as in human Parkinson's disease itself. In addition to ablation, implantation of electronic stimulators are commonly employed. The mechanism of the stimulators is believed to be mediated by local inhibition (via GABA signaling), and is replicated by the local infusion of GABA agonists.
Like Parkinson's disease, methods for treating epilepsy include the use of anti-epileptic drugs, such as sodium valporate (Epilim). Available drugs reduce seizure frequency in the majority of patients, but it is estimated that only about forty percent are free of seizures despite optimal treatment. Other forms of treatment include DBS of certain regions of the brain, such as the VIM (ventral intermediate thalamus), subthalamic nucleus, and internal globus pallidus. However, the DBS procedure is not always effective in many patients who require repeated treatment.
Each of these approaches, surgical ablation, electrical stimulation and infusion of pharmacological GABA agonists is effective in disease palliation, but each has significant adverse effects. For example, extensive invasive surgery, a high risk of infection and potential damage to the brain and in the case of drug infusion, very transient efficiency.
Thus, the treatments for neurodegenerative disorders are palliative at best, with limited and transient efficacy. Therefore, a need exists for a therapeutic approach which has advantages in targeting specificity, both short and long-term efficacy, as well as neuroprotection, without extensive surgery or side-effects.